Effects of N-Terminal Deletion Mutation on Rabbit Muscle Lactate Dehydrogenase

Yanbin Zheng¹, Shuyuan Guo¹, Zhi Guo¹, and Xicheng Wang^{1,2See footnote 1}

¹Department of Biological Science and Biotechnology, Tsinghua University, Beijing 100084, China; fax: 8610-6277-2248; E-mail: wangxic@mail.tsinghua.edu.cn

²Protein Science Laboratory of the Ministry of Education, School of Life Science and Engineering, Tsinghua University, Beijing 100084, China

^* To whom correspondence should be addressed.

Received May 26, 2003; Revision received June 20, 2003

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Deletion mutants of rabbit muscle lactate dehydrogenase (LDH) were constructed using polymerase chain reaction (PCR) to study the roles of N-terminal residues. The coding sequences of the first 5 (LD5) and 10 (LD10) amino acids of the N-terminus were deleted and the gene was inserted into the prokaryotic expression vector pET21b. The mutant enzymes were expressed in E. coli BL21/DE3 and were purified. Then their characteristics and stabilities were studied. The results showed LDH was completely inactivated when the first 10 N-terminal amino acid residues were removed, but the mutant (LD10) could have partially restored activity in the presence of structure-making ions. The removal of the first 5 and 10 N-terminal amino acid residues did not affect the aggregation state of the enzyme, that is, LD5 and LD10 were still tetramers. The stabilities of recombinant wild-type LDH (RW-LD), LD5, and LD10 were compared by incubating them at low pH, elevated temperature, and high GuHCl. The results showed that the N-terminal deletion mutants were more sensitive to denaturing environments; they were easily inactivated and unfolded. Their instability increased and their ability to refold decreased with the increased number of amino acid residues removed from the N-terminus of LDH. These results confirm that the N-terminus of LDH plays a crucial role in stabilizing the structure and in maintaining the function of the enzyme.

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KEY WORDS: lactate dehydrogenase, deletion mutation, stability

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